Current Students with Active Fellowships
Brett Bell NIH NRSA F30 Fellowship for a project entitled "Anti-Complement Immunotherapy for Pancreatic Cancer" (Sponsor, Chandan Guha, Pathology)
Erik Guillen NIH NRSA F31 Fellowship for a project entitled "Impact of T cell receptor signaling on memory CD8+ T cell stemness" (Sponsor, Gregoire Lauvau, Microbiology & Immunology)
Helen Jung NIH NRSA F30 Fellowship for a project entitled "Strategies for next-generation flavivirus vaccine development " (Sponsor, Jon Lai, Biochemistry)
Riana Lo Bu NIH NRSA F31 Fellowship for a project entitled " Dissecting GWAS Identified Risk Variants in Parkinson's Disease – Functional Role of GPNMB in the Pathogenesis of PD " (Sponsor, Frank Soldner, Neuroscience)
Vanessa Ruiz NIH NRSA F31 Fellowship for a project entitled "Characterizing subsets of HIV-infected and uninfected CD14+CD16+ monocytes that contribute to neuropathogenesis" (Sponsor, Joan Berman, Pathology)
Jessie Larios Valencia NIH NRSA F31 Fellowship for a project entitled "The Role of Dedifferentiation in Basal like Breast Cancer" (Sponsor, Wenjun Guo, Cell Biology)
Eric Sosa NIH NRSA F31 Fellowship for a project entitled "Defining the gene regulatory roles of non-coding variants in the pathogenesis of autism" (Sponsor, John Greally, Genetics)
Tram Nguyen NIH NRSA F30 Fellowship for a project entitled "Reward Function in Adolescents with Depression and Cannabis Use" (co-Sponsors, Vilma Gabbay and Benjamin Ely, PCI-Neuroscience & Psychiatry)
Gabriel Bedard NIH NRSA F30 Fellowship for a project entitled "Rational design of anti-cancer therapeutics harnessing the synthetic lethality of methionine metabolism and arginine methyltransferases" (Sponsor, Vern Schramm, Biochemistry)
Matanel Yheskel NIH NRSA F31 Fellowship for a project entitled "Epigenetic and transcriptional consequences of Intellectual Disability-associated mutations in the histone lysine demethylase KDM5" (Sponsor, Julie Secombe, Genetics)
Andrea Bae NIH NRSA F30 Fellowship for a project entitled "Role of brain oscillations in midbrain and forebrain networks supporting stimulus selection in the sound localization pathway of barn owls" (Sponsor, Jose Luis Pena, Neuroscience)
Jacob Stauber NIH NRSA F30 Fellowship for a project entitled "Understanding stem-cell evolution dynamics of donor clonal hematopoiesis in allogeneic hematopoietic cell transplantation at a single-cell level" (co-Sponsors, John Greally and Ulrich Steidl, Genetics and Cell Biology)
Leti Nunez NIH NRSA F31 Fellowship for a project entitled "Determining the effect of RNA binding protein phosphorylation on mRNA fate" (Sponsor, Robert Singer, Anatomy and Structural Biology)
Chris Nishimura NIH NRSA F30 Fellowship for a project entitled "Mechanistic Dissection and Therapeutic Targeting of B7x in Cancer" (Sponsor, XingXing Zang, Microbiology & Immunology)
John "Jack" Barbaro NIH NRSA F30 Fellowship for a project entitled "Methamphetamine and Antiretroviral Therapy Impact Macrophage Functions and Macroautophagy: Implications for HIV Neuropathogenesis" (Sponsor, Joan Berman, Pathology)
Ryan Graff NIH NRSA F30 Fellowship for a project entitled "Platelet PI3Kβ regulation of metastasis" (Sponsor, Jonathan Backer and Anne Bresnick, Molecular Pharmacology)
Daniel Borger NIH NRSA F30 Fellowship for a project entitled "Developing a novel ex vivo platform to support hematopoietic cells and characterize the stem cell niche" (Sponsor, Paul Frenette, Cell Biology)
Bianca Ulloa NIH NRSA F31 Fellowship for a project entitled "Deciphering the development of hematopoietic stem and progenitor cell self-renewal and differentiation" (Sponsor, Teresa Bowman, Developmental & Molecular Biology)
Taylor Thompson NIH NRSA F31 Fellowship for a project entitled "Transcriptional Regulatory and Cell Differentiation Influences of an Endocrine Disrupting Chemical" (Sponsor, John Greally, Genetics)
Current Students with Completed Fellowships
Ian MacArthur NIH NRSA F30 Fellowship for a project entitled "Epigenetic regulation of neural stem cell biology by Tet DNA dioxygenases" (Sponsor, Meelad Dawlaty, Genetics)
Henrietta Bains NIH NRSA F31 Fellowship for a project entitled "How does mTOR sense lipid in vivo" (Sponsor, Rajat Singh, Developmental & Molecular Biology)
Julio Flores NIH NRSA F31 Fellowship for a project entitled "Epigenetic regulation of stem cells and development by the DNA dioxygenase Te2" (Sponsor, Meelad Dawlaty, Genetics)
Michelle Gulfo NIH NRSA F31 Fellowship for a project entitled "Assessing dopaminergic modulation of an associative circuit within the dentate gyrus" (Sponsor, Pablo Castillo, Neuroscience)
Meera Trivedi NIH NRSA F31 Fellowship for a project entitled "Characterizing Novel Regulations of Dendritic Tiling in C. elegans" (Sponsor, Hannes Buelow, Neuroscience)
Hayden Hatch NIH NRSA F31 Fellowship for a project entitled "Transcriptional regulation, neuronal development, and function of the mushroom body in a Drosophila model of intellectual disability" (Co-Sponsors, Julie Secombe and Nicholas Baker, Neuroscience/Genetics)
Taylor Thompson NIH NRSA F31 Fellowship for a project entitled "Transcriptional Regulatory and Cell Differentiation Influences of an Endocrine Disrupting Chemical" (Sponsor, John Greally, Genetics)
Abstracts of Recent Projects
Eric Sosa - ABSTRACT: In this Predoctoral Fellowship proposal, I will be trained for a future as a physician-scientist with my own independent research program at the interface of genomics, computational biology, and neuroscience. During my MD/PhD training, I will be co-mentored by two physician-scientists, Drs. John Greally (Medical Genomics) and Pablo Castillo (Neurology), addressing a question that is timely with the imminent widespread use of whole genome sequencing (WGS) in clinical diagnostics – how do we interpret variants in the noncoding majority of the human genome when a patient presents with a medical problem? I will focus on autism, as a condition that represents a substantial proportion of patients seen by medical genetics services, for which there is extensive WGS information from thousands of families. Despite this wealth of research sequencing, only a small minority of individuals with autism receive a positive outcome of diagnostic exome or WGS. I propose that the currently limited diagnostic success rates are mostly due to our inability to interpret pathogenic variants in the non-coding majority of the genome of these patients. By improving our insights into non-coding variants, we will be able to offer diagnostic information to many more families seeking answers than currently. My strategy is to focus on de novo variants (DNVs) in offspring with autism born to unaffected parents. My hypothesis is that DNVs can be pathogenic when they occur in the cis-regulatory regions of cell types mediating autism. The project is therefore based on a computational genomics foundation, using WGS and DNV calls from large datasets from thousands of families who have a member with autism. In my preliminary data, I show that DNVs in individuals with autism are enriched at cis-regulatory loci in glial and neuronal cells in particular, and at genes known to be causative for autism. In my project, I will test these associations more rigorously, and will define a high-confidence set of DNVs for functional testing. Two types of functional testing will be performed. One will test whether the DNVs alter molecular genomic properties, including chromatin accessibility and gene expression. The second will test the physiological properties of the cells. To generate the appropriate cells for testing, I plan to use induced pluripotent stem cells (iPSCs) that are in vitro differentiated to GABAergic neurons and astrocytes. By performing CRISPR-mediated genomic editing in the iPSCs, I can generate cells with the candidate pathogenic DNVs, and test whether they have effects on cellular properties like dendritogenesis, synaptogenesis, and electrophysiology, increasing the confidence that these DNVs have pathogenic effects. I will have the privilege of getting training in sophisticated computational, stem cell and neuroscience techniques, under the guidance of two leaders in their fields, as part of a comprehensive training plan that will equip me to become the independent physician-scientist I aspire to be in my career.
Tram Nguyen - ABSTRACT: Adolescent depression and cannabis use are concurrent major public health concerns, and this comorbidity has been associated with long-term cognitive and behavioral consequences. However, there has been sparse research in this area, as most neuroimaging studies in adolescent depression exclude cannabis users. We seek to address this gap based on converging evidence that: 1) Reward dysfunction contributes to maintenance and progression of depression in adolescents; 2) Reward dysfunction is a heterogenous construct involving deficits in diverse cognitive processes, including reward anticipation, attainment, and prediction error; 3) These deficits entail distinct neural mechanisms that can be studied by functional magnetic resonance imaging (fMRI); 4) The major psychoactive agent in cannabis, Δ9-tetrahydrocannabinol (THC), exerts its effects through modulation of the cortico-striatal reward system; and 5) Cannabis use may result in temporary relief of mood and anxiety symptoms while inducing potentially deleterious long-term neural reward alterations. We documented that anhedonia–a core symptom of depression reflecting reward deficits–was associated with worse depression outcomes, including chronicity and suicidality, in adolescents. Similarly, using resting-state fMRI, we found that altered striatal intrinsic functional connectivity (iFC) with the prefrontal cortex was associated with anhedonia severity in depressed adolescents. Further, using the reward flanker task (RFT), we found that adolescents with diverse mood and anxiety symptoms showed weaker striatal activation during reward anticipation than healthy controls, while stronger activation in the anterior cingulate cortex predicted worse higher levels of anhedonia at two-year follow-up. We therefore propose a 2×2 cross-sectional study to test the overall hypothesis that comorbid cannabis use in adolescent depression is associated with more severe neural reward deficits and clinical symptoms. Participants will comprise 30 depressed cannabis users, 30 depressed cannabis non-users, 30 non-depressed cannabis users, and 30 healthy controls, all ages 14-18 (Tanner stage ≥ 4) and psychotropic-medication-free. Using resting-state and RFT task fMRI, we predict that comorbid cannabis use and depression will be associated with more pronounced deficits in reward anticipation (Aim 1) and alterations in cortico-striatal iFC (Aim 2) than depression alone. Further analyses (Exploratory Aims) will assess the relationships between cannabis use frequency and activation during reward anticipation, cortico-striatal iFC, and symptom severity. Public Health Relevance: This proposal aims to address a critical and growing public health concern that to date has received minimal research attention while providing a rigorous framework for predoctoral training in both clinical research and advanced neuroimaging techniques. Findings from this work will expand the limited understanding regarding the impact of cannabis on reward dysfunction in youth, with potential to inform both clinical practice and government policies on cannabis use.
Gabriel Bedard - ABSTRACT: Methionine adenosyltransferase 2 alpha (MAT2A) and protein arginine methyltransferase 5 (PRMT5) are cancer targets that are synthetically lethal with MTAP deletions and have several drug candidates in clinical trials targeting MTAP-/- cancers. MTAP is deleted in ~15% of human cancers and encodes the metabolic enzyme 5’-methylthioadenosine phosphorylase, the sole enzyme in humans responsible for recycling of methylthioadenosine (MTA) to methionine. MAT2A synthesizes S-adenosyl methionine (SAM), the methyl donor substrate for methyltransferase reactions. PRMT5 utilizes SAM as a substrate and is inhibited by MTA, and MTAP-/- cells in culture demonstrate elevated MTA levels. In vivo observations of glioblastoma tumors suggest however, that MTAP-/- does not always lead to increased tumoral MTA levels due to MTA efflux into matrix MTAP-competent cells. Additionally, MTAP deletions are a rare (~2%) occurrence in colorectal cancers (CRCs), precluding MAT2A and PRMT5 inhibitors’ use for most CRCs. The Schramm laboratory has previously solved the transition state (TS) structure of MTAP and synthesized a potent small molecule inhibitor methylthio-DADMe-immucillin-A (MTDIA) that recapitulates the in vitro effects of MTA accumulation within tissues. MTDIA has been shown to inhibit tumor growth in several cancer models, including CRC, and is linked to a decrease in PRMT5 activity through elevation of MTA levels. We propose that MTDIA be used in combination with MAT2A inhibitor AG-270, currently in Phase I clinical trials, to harness their synthetic lethality by targeting PRMT5. We will test the safety, target engagement, and anti-cancer efficacy of MTDIA in combination with AG-270 in ApcMin/+ and CRC patient-derived xenograft (PDX) mice. To determine mechanisms of anti-cancer effects, we will probe the upstream and downstream effects related to PRMT5 activity. We will perform tumor metabolomic quantification of relevant metabolites and histone and protein-arginine methylation characterization using immunohistochemistry and proteomic techniques. We will also profile the gene expression changes using single-cell RNA sequencing to determine how combination therapy alters tumor architecture and growth. Finally, we will solve the transition state structure of PRMT5 with the goal of laying the foundations for development of novel transition state analogue inhibitors. This work will expand upon the use of MAT2A and PRMT5 inhibitors beyond the ~15% of MTAP-deleted cancers and provide avenues for MTDIA to be used in clinical trials.
Ian MacArthur - ABSTRACT: The ten-eleven translocation family (TET1/2/3) of enzymes are epigenetic regulators of gene expression that are highly expressed in neural stem cells (NSCs) and during mammalian nervous system development. TET enzymes are dioxygenases that promote active and passive DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher-order oxidized derivatives. In addition to its role as a demethylation intermediate, 5hmC can function as a stable epigenetic mark and is highly enriched and dynamic in the developing nervous system. TET enzymes and 5hmC dysregulation have been implicated in human neurodevelopmental syndromes, intellectual disability, craniofacial abnormalities, and neurodegeneration. These observations suggest a critical role for TET enzymes in the developing nervous system and has led to interest in their roles in the biology of NSCs. However, the functions of TET enzymes in NSCs and neurodevelopment remain poorly understood. Preliminary data from our lab demonstrates that Tet tripleknockout NSCs (T123–/–) derived from embryonic stem cells exhibit severe defects in self-renewal, multipotency, and expression of neurodevelopmental genes. We therefore hypothesize that TET enzymes have essential functions in epigenetic regulation of gene expression programs critical for NSC maintenance and multipotency and in embryonic neurodevelopment. To test this hypothesis, we have derived embryonic forebrain NSC lines containing floxed alleles of Tet1/2/3 and a tamoxifen-inducible Cre recombinase transgene expressed from the constitutive Rosa26 locus (T123F/F; +/R26-CreER) for conditional, combined deletion of all three Tet genes. We have also established a colony of Tet1/2/3 triple-floxed mice expressing a tamoxifen-inducible Cre recombinase transgene under control of the neural-specific Nestin promoter (T123F/F; +/Nestin-CreERT2). Using these models, we will (1) define the role of TET enzymes in the maintenance and multipotency of NSCs , (2) establish the requirement of TETs in embryonic neurodevelopment, and (3) identify TET-mediated epigenetic and transcriptional regulatory mechanisms in NSCs. Findings from these studies will define novel roles played by TET enzymes in NSC biology and neurodevelopment, provide insights into how dysregulation of TETs contribute to human neurodevelopmental disorders, and identify novel targets for therapy.
Chris Nishimura - ABSTRACT: Immune checkpoints are proteins that regulate the body’s immune system via inhibition of immune cells. In diseases such as cancer, these same pathways may be commandeered in order to inappropriately inhibit immune responses. One approach to overcome this excessive inhibition and restore normal immune function is to physically block these inhibitory proteins using monoclonal antibodies (mAbs), a strategy known as immune checkpoint blockade. Our lab discovered the protein B7x (VTCN1/B7S1/B7-H4), an immune checkpoint whose regulation and mechanism of action are still being elucidated. B7x is expressed on a wide variety of cancers and is associated with poor clinical outcomes. It has been shown to inhibit effector T cell functions such as cytokine production and proliferation, and promotes T cell exhaustion; it has also been associated with tumor infiltration of immunosuppressive cell populations such as myeloid derived suppressor cells (MDSCs). Intriguingly, it is not typically co-expressed with the well-studied immune checkpoint PD-L1, and blocking B7x using anti-B7x mAbs improves anti-tumor responses and survival in mouse models. This suggests that B7x holds a non-overlapping but key role in cancer immune evasion. For these reasons I hypothesize that B7x mediates immune evasion through inhibiting effector cell functions while simultaneously promoting immunosuppressive cells and is therefore a promising target for cancer immunotherapy. Thus, we propose the following two aims: 1) Examine the mechanisms that regulate B7x expression and its role on MDSCs; and 2) Develop and characterize a new anti-B7x immunotherapy. In aim 1 I will examine the how hypoxia and other tumor microenvironment-associated cytokines regulate B7x expression. In addition, I will explore how B7x affects the immunosuppressive MDSC cell population generation and survival. In aim 2 I will test the anti-tumor efficacy of newly generated anti-B7x mAb-based immune checkpoint blockade in vivo using metastasis and spontaneous models of lung cancer. We will also characterize how this new immunotherapy alters T cell phenotypes using flow cytometry and single-cell RNA sequencing. Finally, we will explore if combining our anti-B7x mAb with anti-TIM3 and anti-PD1 mAbs confers superior anti-tumor efficacy than anti-B7x monotherapy in vivo. Together, these studies will allow us to broadly explore the role of B7x in tumorigenesis and therapeutic potential of anti-B7x immunotherapy, and combined with a personalized training plan and supportive research environment at Einstein, cultivate the skills required of a physician-scientist.
Ryan Graff - ABSTRACT: Metastasis is the primary cause of morbidity and mortality among patients with solid tumors. Platelets interact with tumor cells upon their entry into the vasculature and promote the metastatic spread of these cells by several mechanisms. Circulating tumor cells (CTCs) directly bind to and activate platelets resulting in the formation of platelet-fibrin complexes that envelope circulating tumor cells and protect them from immune clearance. Adhesion to tumor cells also induces the release of platelet cytokines and other soluble factors that promote epithelial-mesenchymal transition in the tumor cells. Finally, platelets promote the adhesion of CTCs to the endothelium, assisting in their extravasation at distant metastatic sites.
Platelet activation, adhesion, and in vivo thrombus formation require the activity of the Class IA PI 3-kinase PI3K. This isoform of PI3K produces the majority of PI(3,4,5)P3 in platelets, suggesting that PI3K has a unique role in classical platelet activation. However, the role of PI3K in platelet-mediated cancer metastasis has yet to be defined. We propose that PI3K is required for platelet activation upon interaction with tumor cells. Aim 1 will test the role of platelet PI3K in platelet-stimulated tumor cell Matrigel invasion, transendothelial migration, epithelial-mesenchymal transition, cell stemness, NFB activation, and platelet TGF secretion. We will also use RNAseq and antibody arrays to more broadly examine the role of PI3K in platelet chemokine/cytokine release and in transcriptional responses by tumor cells. We will use both platelets from mice expressing mutant PI3K and human platelets pre-treated with the irreversible pan-PI3K inhibitor wortmannin. Aim 2 will directly evaluate the role of platelet PI3K in cancer metastasis in wild type and whole-animal or platelet-specific mutant PI3K mice. Murine mammary carcinoma cells will be injected intravenously to analyze in vivo tumor cell-platelet complex formation as well as tumor cell interactions with macrophages and other leukocytes. We will also test the role of PI3K in tumor cell-induced thrombocytopenia and experimental metastasis.
Isoform selective PI3K inhibitors were originally developed to be used as anti-thrombotic agents, as they prevent thrombotic occlusion of injured arteries without increasing bleeding. More recently these inhibitors have also been explored as anti-cancer agents in the treatment of PTEN-deficient cancers. This proposal seeks to understand the potential for a broader application of these agents in cancer patients to inhibit platelet activity and prevent metastasis in a variety of cancer types.
Henrietta Bains - ABSTRACT: Alterations in lipid metabolism determine metabolic disease and mortality in the aging population. Despite our understanding of regulation of lipid metabolism, how organisms sense lipid remains unknown. It is conceivable that sensing of lipid will inform downstream decisions taken by the cell that modulate metabolism, proteostasis, stress response, and growth—each of which are dysregulated with age. The mechanistic target of rapamycin (mTOR), is a serine/threonine kinase and amino acid sensor, that drives growth and proliferation. More recently, mTOR in cultured cells has been shown to be activated by cholesterol and phosphatidic acid (PA) in absence of amino acids. Whether mTOR senses lipid in whole organisms is unclear. mTOR exists as two major complexes—mTORC1 and mTORC2. Activation of mTORC1 occurs at the lysosomal surface in presence of amino acids and requires key regulatory proteins that stimulate its activity. By contrast, mTORC2 responds to growth factors to regulate cytoskeletal organization. Hyperactivation of mTORC1 (hereafter, mTOR for simplicity) drives aging and age-related diseases in part by disrupting autophagy and promoting growth. Indeed, suppressing mTOR has been shown to increase lifespan in multiple organisms. However, how mTORC1 is hyperactivated with age remains unknown. It has been shown that there are quantitative and qualitative changes in membrane lipids with age including changes in lysosomal membrane lipids—the major site of mTOR activation. Whether changes in lysosomal membrane lipids are mechanistically linked to mTOR hyperactivation remain unknown. Our preliminary data show that subjecting mice to an oral gavage of corn oil causes activation of mTOR and its translocation to distinct cholesterol-rich microdomains (CRMs)/lipid rafts in lysosome membranes. Our preliminary data also show that immunoprecipitating mTOR from lysosome membranes from livers of oil-gavaged mice reveal its binding to diacylglycerol. These data suggest that mTOR is a sensor of diacylglycerol, a membrane lipid. Since mTOR senses nutrients at lysosome membranes, I hypothesize that mTOR senses lipid at lysosomal membranes, and that age-related changes in lysosomal membrane lipid composition lead to mTOR hyperactivation. To test our hypothesis, we present the following specific aims: Aim 1: To determine how mTOR senses lipid at the lysosome surface. In Aim 1, diverse approaches will be used to characterize lipid-driven mTOR activation at lysosome membranes. By pulling down mTOR from lysosome membranes for lipidomic and proteomic analyses, I will identify lipid species that bind to mTOR and its interacting partners. I will use an siRNA screen in vitro to silence each of the interacting partners, which will identify novel regulators of lipid-driven mTOR signaling. Aim 2: To determine the mechanism of mTOR hyperactivation with age. In Aim 2, I will characterize the changes in lipid composition of lysosome membrane CRMs and expansion of lysosome CRMs with age. I will determine whether alterations in membrane lipid composition with age correlate with increased mTOR activity. I will then determine whether inactivating the synthesis of specific membrane lipids, e.g., PA and DG, by shRNAs against relevant biosynthetic enzymes in liver will dampen age-related mTOR hyperactivation. I will also determine whether targeting key interacting partners of mTOR in liver will dampen age-related hyperactivation of mTOR signaling and reverse deleterious mTOR-dependent outcomes, i.e., blockage of autophagy and proteostasis failure.
Julio Flores- ABSTRACT: The Ten-eleven translocation (Tet1/2/3) family of enzymes are epigenetic regulators of gene expression important for stem cell biology and embryonic development. Tet enzymes are dioxygenases that promote DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher oxidized derivatives. In addition to this enzymatic activity, Tet enzymes can bind chromatin modifying complexes, to regulate genes in a presumably catalytic-independent manner. Tet2 is a key member of this family. It is highly expressed in embryonic stem cells (ESCs) and controls gene expression programs necessary for stem cell lineage specification. Tet2 is also frequently mutated in hematological malignancies and has been implicated in neurodegenerative diseases. While the catalytic functions of Tet2 have been well studied, its non-catalytic roles remain poorly defined. In this proposal, we seek to establish the significance of the catalytic dependent and independent functions of Tet2 in ESC gene regulation and lineage commitment. We hypothesize that Tet2, in addition to regulating genes through its DNA demethylase activity, can also modulate genes in a non-catalytic fashion by recruiting histone modifiers to the chromatin, and this dual mode of gene regulation is essential for proper ESC differentiation along the neural and hematopoietic lineages. To test this hypothesis, I have generated Tet2 catalytic mutant (Tet2m/m) and knock-out (Tet2–/–) ESCs, which I will use as a platform to: (1) identify the catalytic and non-catalytic direct target genes of Tet2 in ESCs by integrating changes in gene expression with Tet2 genomic occupancy, (2) establish Tet2-mediated activating and repressing mechanisms of gene regulation involving interactions with histone modifiers OGT and HDAC2, and finally (3) define the biological significance of Tet2 enzymatic and non-enzymatic functions in ESC differentiation and lineage commitment along the neural and hematopoietic lineages. Findings from these experiments will elucidate novel epigenetic mechanisms of gene regulation in ESCs involving Tet2 catalytic and non-catalytic functions. They will enhance our understanding of stem cell biology and development and can have implications in hematological malignancies where Tet2 is affected.
Daniel Borger- ABSTRACT: Current culture methods reduce the ability of hematopoietic stem cells (HSCs) to successfully engraft in a host. Emerging gene editing technologies such as CRISPR/Cas9 require time in culture to allow for the correction of disease-causing alleles. There is therefore a need to develop new methods of culturing HSCs. Coculture of HSCs with bone marrow niche cells such as mesenchymal stem cells (MSCs) is one possible solution to problems in HSC culture, as these cells provide factors that support HSCs in vivo. However, MSCs cannot be maintained in culture for extended periods of time and fairly rapidly lose expression of niche factors. Through a screen of candidate transcription factors, our lab identified 5 factors that when transduced together restore niche factor expression and allow for prolonged culture. These factors are Kruppel-like factor 7 (Klf7), Osteoclast stimulating factor (Ostf1), X-box binding protein (XBP1), Interferon regulatory factor 3 (Irf3), and Irf7, which we collectively dubbed the KOXII factors. KOXII-transduced MSCs were able to expand both murine and human funtional HSCs to a much greater extent than mock-transduced MSCs. These cells therefore may be useful in expanding HSCs ex vivo for gene correction. However, there are regulatory barriers to the application of murine cells in human therapeutics. The work proposed here will in part focus on the development and characterization of KOXII-transduced human MSCs. After generating these cells, I will determine if the KOXII factors affect expression of niche factors in human MSCs. I will also use flow cytometry and stem cell xenotransplantation to determine if KOXII-transduced MSCs are more effective at driving HSC expansion than unmodified MSCs. Finally, using CRISPR/Cas9-based gene editing of HSCs derived from patients with sickle cell disease as a model, I will determine if coculture of patient cells with KOXII-transduced MSCs can improve the efficiency of gene editing or increase the yield of properly edited cells over current standard HSC culture methods. In parallel, I will use murine KOXII-transduced MSCs to more closely examine niche signalling by MSCs. As these cells can be cultured in relatively large numbers, they are ideal for proteomic studies. In collaboration with the lab of Jeroen Krijgsveld, I will examine the secretome of these cells in order to identify proteins whose secretion is upregulated by the KOXII factors. Using both in vitro and in vivo assays, I will evaluate the effect of these factors on HSC maintenance and proliferation, with the aim of identifying secreted proteins with previously unappreciated roles in MSC-HSC niche interactions.
Ryan Malonis - ABSTRACT: Alphaviruses are enveloped, positive sense single-stranded RNA viruses, which include several important human pathogens. Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause human rheumatic disease and include chikungunya virus (CHIKV) and Mayaro virus (MAYV). Symptomatic infection is characterized by fever, rash, myalgia, as well as both acute and chronic polyarthralgia that can persist for months to years after infection. More severe manifestations of alphaviral disease – including hemorrhage, encephalopathy and mortality – have been reported. These viruses cause endemic disease as well as large, sporadic epidemics worldwide. Currently, there are no approved vaccines or anti-viral therapies for the prevention or treatment of alphavirus infection; therefore, the development of new therapeutic strategies targeting one or multiple arthritogenic alphaviruses is of substantial interest. A number of potently neutralizing CHIKV monoclonal antibodies (mAbs) have been described, but currently the only broadly neutralizing alphavirus mAbs that have been reported are murine. Thus, the extent to which the human antibody response elicits broadly-neutralizing mAbs following alphavirus infection, and which epitope(s) such mAbs may target, remains unknown. To address this question, this proposal seeks to expand our knowledge of the neutralizing antibody response to alphaviruses by systematically investigating cross-reactive antibodies from CHIKV-infected patients. Towards this end, we have used single B cell sorting to isolate a large panel of MAYV-reactive mAbs from CHIKV patients in the convalescent phase. We will study the reactivity and neutralization profiles of these mAbs against related arthitogenic alphaviruses (Aim 1). We will then biochemically determine the requirements of neutralization (Aim 2) and elucidate the mechanism of mAb inhibition (Aim 3). These studies will contribute to our fundamental understanding of how the adaptive immune system combats infection by arthritogenic alphaviruses and may aid the development of novel mAb-based treatments and vaccines.
Bianca Ulloa - ABSTRACT: Hematopoietic stem and progenitor cells (HSPCs) are characterized by their self-renewal and multipotent differentiation capacities. As such, they give rise to all mature blood cell types (e.g., myeloid, lymphoid, and erythroid) to maintain life-long hematopoiesis. Their regenerative capacity makes HSPCs valuable for cell replacement therapies in patients with hematological diseases, including those that are secondary to chemotherapy and radiotherapy. Understanding HSPC properties of self-renewal and multipotency allows for the development of methods to improve and maximize their therapeutic potential. By studying the embryonic origins of HSPC self-renewal and differentiation capacities, we aim to advance what is known about these defining characteristics of stem cells. In addition to HSPCs, other multi-lineage progenitors are produced during embryogenesis. These are limited in their self-renewal and differentiation output and are mostly regarded as transient in nature. These progenitor populations share many features of stem cells, confounding studies of HSPC properties within their native embryonic environments. Several studies in murine and zebrafish models suggest that these embryonic progenitors, and not HSPCs, are the dominant population generating mature blood cells in the embryo. If HSPCs are not necessary to sustain the embryo, what is their function during development? To answer this question, we propose to use zebrafish to determine when and where during development HSPCs self-renew and contribute to mature blood cell output in myeloid, lymphoid, and erythroid lineages. We will use novel regeneration and transplantation assays (Aim 1) to study self- renewal and lineage-tracing experiments (Aim 2) to investigate HSPC differentiation. Understanding how these properties are established and maintained is critical to harnessing stem cells for regenerative medicine.
Taylor Thompson - ABSTRACT: One major group of environmental toxicant that affect humans negatively are endocrine disrupting chemicals (EDCs). These chemicals interfere with the body’s natural hormone regulation leading to a range of human diseases. Our research focuses on the EDC tributyltin (TBT), a chemical frequently used as a pesticide and plastic stabilizer. TBT has major adipogenic effects when exposed in utero or in adult multipotent stem cells. Previously published data have demonstrated that TBT exposure promotes differentiation of mesenchymal stem cells (MSCs) into adipogenesis, and also increases their lipid content, representing both numerical and qualitative effects on adipocytes. Mechanistically, TBT has been found to bind to the ligand-binding domain of the peroxisome proliferator-activated receptor gamma (PPARg) transcription factor (TF), which is known to form a heterodimer with the RXR TF when activated, promoting a transcriptional reprogramming of MSCs to commit them to adipogenesis. MSCs can differentiate into a number of lineages, including muscle, bone, cartilage and fibrocystic cells. When a cell undergoes transcriptional reprogramming, the sites at which the TFs bind change, reflected by alterations of the distributions of loci of open chromatin. In this project, we propose to differentiate MSCs to both adipocytes and myocytes, initially using the cell culture conditions known to promote specific differentiation of MSCs. We will map the loci of open chromatin and test gene expression in these samples, allowing us to identify TFs mediating these differentiation pathways by searching for motif enrichment corresponding to known TF binding sites. With this information available, we can then use the same approaches to test how TBT causes transcriptional reprogramming, which should reveal whether the process is identical or involves a different set of TFs. Finally, we will apply the new CellTagging approach to test cells at multiple stages of differentiation to myocytes to test whether TBT exposure affects only undifferentiated MSCs, or can also cause transdifferentiation of cells already developing in the myogenic lineage. These new insights into the mechanism of action of TBT will be valuable in understanding how EDCs have their disease- causing effects. We will also get insights from TBT into how we a small molecule can mediate ‘epigenetic therapy’, influencing transcriptional reprogramming but in a way that is targeted to specific genomic locations. Under the mentorship of Drs. John Greally and Paul Frenette, I will accomplish these goals while developing new skills in developmental biology and genetics. Additionally I will gain valuable experiences in presenting, networking, and manuscript writing, all of which are essential as I train to become and independent investigator and physician-scientist.